1. Field of the Invention
The present invention relates to a method for preparing human neoplastically transformed cells. More specifically, the present invention relates to a method for preparing neoplastically transformed cells from human-derived cells using antisense RNA derived from human 28S rRNA, cells obtained by the method, a kit for use in the method, and a method of using the cells obtained by the method in assessment of pharmacologically efficacy of an anticancer agent.
2. Discussion of the Related Art
From the studies of human-derived cells using an oncogene encoding a DNA tumor virus protein, such as a Simian virus 40 (SV40) large T antigen, an adenovirus E1A or E1B, Human papillomavirus E6 or E7, a model of carcinogenesis presumes that malignant transformation of human cultured cells consists of two phases of an immortalization process and a tumorigenetic process.
In the immortalization process, telomeric sequences are added to end parts of chromosomes by telomerase activity, thereby making it possible to acquire an infinite lifespan. Therefore, if immortalization takes place, a clonal expansion of cells takes place, so that opportunity for additional genetic alterations is increased, thereby increasing a possibility of tumorigenetic conversion. In addition, in the tumorigenetic process, the immortally transformed cells acquire an autonomous growth potential in the absence of certain growth factors.
Generally, cancer cells express uncertain phenotypes different from normal cells, e.g. genomic instability, lack of responsiveness to the chemical signalings, and loss of differentiation. However, it yet remains obscure whether those alterations involved in the tumorigenetic process described above result from cumulative mutations in genes related to the particular proteins, or a cascade of gene expression initiated by a factor activated in the onset of tumorigenesis about which little is known.
In recent years, it has been reported that normal human cells can be transformed by expression of human telomerase catalytic subunit (hTERT), SV40 early region (ER), and activated H-ras gene (See W. C. Hahn et al, “Creation of human tumour cells with defined genetic elements.” letter, Nature, 29 Jul. 1999). According to the report, in the presence of hTERT, the SV40 large T antigen (LT) inactivates retinoblastoma protein (pRB) tumor suppressor pathway and p53 tumor suppressor pathway, and the SV40 small T antigen (ST) serves to dysfunction phosphatase 2A. Also, the activated ras gene is known to participate in the tumorigenecity of transformed cells. Therefore, the aberrations are suggested to be the minimal number of genetic events required in the malignant conversion of human-derived cells.
In addition, Japanese Patent Laid-Open No. 2008-109889 discloses a method for inducing neoplastic transformation of human-derived cells, including expressing Src in the presence of hTERT and an SV40 large T antigen (LT).
On the other hand, the present inventors have previously reported that a nucleotide sequence resulting from addition of a poly(A) chain to a certain nucleotide sequence at a 3′-end side of a first stem of a secondary structure of U5 small nuclear RNA [also named as transforming RNA (TR)] is expressed as non-coding mRNA, thereby making it possible to drive rat fibroblastic 3Y1 cells into the neoplastically transformed cells, i.e. tumor cells (See K. Hamada, “Morphological transformation caused by a partial sequence of U5 small nuclear RNA.” Mol. Carcinog., 1997, 20, 175-188). The 3Y1 cells had already acquired the ability to proliferate infinitely through spontaneous immortalization. Therefore, it is deduced that the TR plays a role in tumorigenesis, not in immortalization; as a result of further studies in view of the above, the present inventors have found that the above-mentioned transformation is greatly dependent on a certain polypurine sequence ggagaggaa (SEQ ID NO: 3) of the TR.
Furthermore, it is reported that as a result of studies using a rabbit reticulocyte extract, the TR sequence synthesized in vitro directly binds to 28S rRNA of ribosome in the nascent chain elongation of peptide synthesis to affect secretory signal peptide-associated translation (K. Hamada et al, “Effect of Transforming RNA on the Synthesis of a Protein with a Secretory Signal Sequence in Vitro.” J. Biol. Chem., 1999, 274(22), 15786-15796). Moreover, it is found that the oligodeoxynucleotides (ODNs) containing the above-mentioned polypurine sequence bind to cucc sequence (SEQ ID NO: 4) on 28S rRNA, and on the other hand that the oligodeoxynucleotides (ODNs) containing an antisense sequence of the above polypurine sequence bind to gagg (nucleotides 48 to 51) (SEQ ID NO: 5) on 7SL RNA of signal recognition particle (SRP). In view of the above, it is considered that the chain elongation arrest activity influences physiological actions between the 28S rRNA and the SRP RNA.
However, it has been found that the above-mentioned TR cannot be induced into neoplastically transformed cells by transforming human-derived cells in the presence of hTERT, depending upon a combination with c-myc, an activated H-ras, or a gene encoding SV40 Large T or the like.
An object of the present invention is to provide a method for preparing neoplastically transformed cells from human-derived cells, cells obtained by the method, a kit for use in the method, and a method of using cells obtained by the method in assessment of pharmacological efficacy of an anticancer agent, which are free from any safety problems.
These and other objects of the present invention will be apparent from the following description.